CN114929386A - Asymmetric size-based nanopore membrane (ANM) filtration for efficient exosome separation, concentration and fractionation - Google Patents

Abstract

Described herein is a size-based Asymmetric Nanopore Membrane (ANM) filtration technique for efficient exosome separation, concentration and fractionation. This ANM design prevents exosomes from deforming, lysing and fusing due to strong external forces, thereby significantly increasing yield (up to 92%), while retaining other advantages of size-based ultrafiltration. It also provides a unique feature that enables washing contaminating proteins from exosomes. It provides higher throughput, yield, sample purity, concentration factor, and more accurate size fractionation than current methods.

Description

Asymmetric size-based nanopore membrane (ANM) filtration for efficient exosome separation, concentration and fractionation

Cross reference to related applications

This application claims priority to U.S. provisional patent application No. 62/901,117, filed on 2019, month 9, and day 16, which is incorporated herein by reference in its entirety.

Federally sponsored research

The invention was made with U.S. government support under grant numbers 1R21CA206904-01 and HG009010-01 of the national institutes of health. The united states government has certain rights in this invention.

Technical Field

Described herein is a size-based Asymmetric Nanopore Membrane (ANM) filtration technique for efficient exosome separation, concentration and fractionation. This ANM design prevents exosomes from deforming, lysing and fusing due to strong external forces, thereby significantly increasing yield (up to 90%), while retaining other advantages of size-based ultrafiltration. It also provides a unique feature that enables washing contaminating proteins from exosomes. It provides higher throughput, yield, sample purity, concentration factor, and more accurate size fractionation than current methods.

Background

Liquid biopsy and other disease screening techniques are based on quantification of nucleic acid and protein biomarkers in blood. It is now recognized that such molecular biomarkers are encapsulated in nanoscale particles such as microvesicles (mv), exosomes (ex), exosomes (exemeres), High Density Lipoproteins (HDL), low density Lipoproteins (LD), and Ribonucleoproteins (RBP). Some nanoparticles are vesicular and some are protein-RNA complexes. Different particles originate from different cells, and nanoparticles (RNPs) containing nitroxyl radicals only appear when the cells are under oxidative or mechanical stress. Some particles have surface proteins inherited from the cells from which they are derived. This heterogeneity of molecular carriers means that accurate diagnosis of disease requires not only quantification of nucleic acid and protein cargo, but also separation and fractionation of nanoparticles. Also, the isolation of nanoparticles from cell cultures (cell culture medium/supernatant) for biomarker discovery, drug testing, drug delivery and cosmetic purposes must be properly fractionated and purified so that only nanoparticles with the proper molecular load are used. Nanoparticles range from 20nm to 300nm, which makes them extremely difficult to rank at the high throughput required for diagnostic, biomarker discovery, drug testing and delivery applications. Ultracentrifugation, precipitation, size exclusion chromatography and nanofiltration enable high-throughput fractionation only if the suspension is forced through a nanofilter using large centrifugal and high pressure/shear forces. The result is particle loss due to cracking. This is disadvantageous for the diagnosis, since an accurate quantification is now not possible.

Exosomes are membrane-encapsulated vesicles (extracellular vesicles) secreted in all living cells, with diameters of 50 to 200nm [ 1]]. Free exosomes are produced by endosomogenous multivesicular bodies (MVBs) released during fusion with the plasma membrane. Most importantly, the exosomes carry mRNA, miRNA and protein derived from the cell from which they are derived [2-5]. The proliferation of exosome-related studies was partly attributed to the Swedish scientist Jan at the University of Gothenburg

Exosomes have long been viewed as tiny garbage sacs thrown out of cells only, but

In 2007, it was shown that some cells use exosomes to transfer genetic material, messenger RNA, between each other to make proteins and microRNAs to regulate gene expression [3]. This finding has prompted scientists to find ways in which exosomes may be involved in health and disease and even used as therapeutics.

The release of microvesicles has been shown to be biologically relevant; these particles act both within the local microenvironment and systemically as mediators of intercellular communication [6 ]. Exosomes are associated with a range of physiological processes including cell proliferation [7], cancer metastasis [8] and immunomodulatory activity [9 ]. In view of these implications, and their presence in clinical samples (plasma, urine, saliva), exosomes represent an emerging target for biomarker discovery with prognostic/diagnostic implications [10 ].

Exosomes have advantages in various aspects compared to other liquid biopsy sources. First, exosomes can reflect primitive cellular markers and even their target cells by presenting specific surface proteins [11 ]. These features allow easy separation of tissue and target cell specific exosomes. Second, exosomes are stable in circulation and are present in all body fluids. They can therefore be used as powerful non-invasive diagnostic tools for a number of diseases including cancer. Third, exosomes exhibit their parental disease/tumor-specific RNA and protein profiles, and their structure protects circulating RNA and microrna (mirna) from rnase catalytic function. Thus, the nucleic acid of exosomes can be used to look for genetic characteristics of cancer patients.

The first blood-based cancer diagnostic product using free-floating exosomes was marketed 21/1/2016, and was developed by Exosome Diagnostics, Cambridge, Massachusetts (www.exosomedx.com /). Exosomal ExoDx Lung (ALK) assay both exosome RNA and ctDNA were detected in a one-step assay. It may improve sensitivity in detecting rare cancer mutations that are not readily detectable in other fluid biopsies relying solely on circulating tumor cells or ctDNA. In a blind study of m0/m1a NSCLC patients whose mutation status was considered particularly difficult to assess by liquid biopsy, the sensitivity of analyzing RNA and ctDNA fractions of exosomes was nearly three-fold improved (74% versus 26%) over that obtained by evaluating ctDNA alone [13 ]. Another key clue in the exosome diagnosis story is pancreatic cancer. In a recent study of 250 patients, Kalluri and colleagues found that glypican-1 (glypican-1, GPC1), a cell membrane-surface proteoglycan, was particularly enriched in circulating exosomes from pancreatic cancer individuals. GPC1 is able to distinguish early and late stage pancreatic cancer from benign disease with 100% accuracy and sensitivity [14 ]. This level of accuracy outperforms any other technique in molecular diagnostics. In addition to exosomes, the broad category of fluid biopsies also includes the analysis of circulating tumor cells and cell-free circulating tumor dna (ctdna) from dying cancer cells. Despite the increasing clinical importance of exosomes as potential biomarkers, current commercial EV isolation methods suffer from low yield and purity, complex procedures and long processing time, and a simple and inexpensive method of isolating circulating exosomes is clinically needed [15 ].

Exosomes are also expected to be a widely used tool for therapy and drug delivery. While liposomes and nanoparticles may provide siRNA delivery advantages over viral-based delivery systems, they exhibit low efficiency and are rapidly cleared from the circulation. Unlike liposomes and other synthetic drug nanoparticle carriers, exosomes contain transmembrane and membrane-anchoring proteins that enhance endocytosis, facilitating delivery of their internal contents [16 ]. Exosome proteins include CD47[17], a widely expressed transmembrane protein associated with integrins whose function is, in part, to protect cells from phagocytosis [18 ]. CD47 is a ligand for signal-regulated protein alpha (sirpa, also known as CD172a), and CD 47-sirpa binding initiates a "do not eat me" signal that inhibits phagocytosis.

A study from the University of Oxford (University of Oxford) Matthew J.A.Wood team showed that exosomes filled with small interfering RNA (siRNA) could reach cells in the mouse brain [19 ]. Once passed through the protective barriers of the brain, genetic material reduces the production of BACE1, BACE1 is a protein involved in alzheimer's disease. In addition, the work of the Raghu Kalluri team at the University of Texas (University of Texas) MD Anderson Cancer Center (MD Anderson Cancer Center) has shown potential to address problems that scientists have been trying to do for over a decade: the siRNA is allowed to enter the correct cells. The team of Kalluri uses engineered exosomes to deliver siRNA that prevent the production of a mutein called KRas, one of the most "drug-free" cancer targets. The siRNA-loaded exosomes of Kalluri injected intravenously inhibited pancreatic cancer in mice better than similar siRNA-loaded lipid nanoparticles injected, and did not have any significant immune response [20 ]. Kalluri is now a joint founder of Codiak Biosciences (www.codiakbio.com) located in massachusetts, one of the more and more biotechnological pioneer companies that attempt to transport drugs by hijacking the exosome messenger system to cells in body parts such as the brain that would otherwise be difficult to reach. However, isolating large numbers of vesicles remains a challenge for exosome-based therapies. Another challenge is how to separate and grade exosomes in view of extracellular vesicle diversity. If all exosomes are of different sizes and therefore the amount of drug loaded is different, the consistency and reproducibility of exosome-based therapy may be compromised.

To facilitate the diagnostic and therapeutic applications of these unique Extracellular Vesicles (EVs), it is crucial to specifically separate exosomes from a broad spectrum of cell debris and interfering components [21 ]. Disease diagnosis often involves quantification of RNA cargo within the exosomes, and treatment requires high yield harvesting of exosomes from the cell culture. Therefore, a technique for isolating exosomes should exhibit high efficiency and be capable of isolating exosomes from various samples. In addition, in view of the diverse extracellular vesicle diversity and the need for downstream analysis and application, separation techniques should be able to concentrate and fractionate exosomes.

Existing commercial technologies take advantage of specific properties of exosomes, such as their density, solubility, size and surface proteins to aid in their separation: ultracentrifugation (UC) is one of the most common techniques for separating exosomes from other EVs of different size and mass, usually requiring a series of centrifugation steps, eventually reaching speeds up to 200000 × g. This technique is time consuming (>4 hours) and provides low exosome recovery (typically < 25%) [22] and low purity, and relatively expensive equipment (>10 ten thousand dollars) due to the presence of non-exosome proteins and microbubble fragments. Density gradient separation is used to purify exosomes by separating exosomes from large proteins. This technique is to extract exosomes from other particles (proteins) by loading the sample onto a concentrated solution of medium (sucrose or inorganic salts) and applying ultracentrifugation based on their different floating densities. Although density gradient separation techniques can improve the purity and recovery of exosomes, they require even longer times (21 hours) than traditional ultracentrifugation and require higher technical capacity from the user [23-24 ].

Precipitation is another commonly used method of exosome isolation. More recently, commercial rapid sedimentation kits such as ExoQuick-TC and Total Exosome Isolation have been able to offer a more affordable (in the $ 200- $ 1000 range) method for many standard hospital laboratories or hospitals in resource-poor countries as compared to centrifugation techniques. The proprietary polymers in these kits gently precipitate exosomes, which are then isolated by centrifugation at lower g-forces, for example, in a typical bench top microfuge (benchtop microfuge). While these methods avoid the problem of equipment cost, long separation times (-24 hours) remain a limiting factor for these technologies. The presence of non-exosome sources such as RNA-binding proteins together with extracted exosomes is a common contaminant that prevents the detection of exosome RNAs of interest.

An alternative to UC is immunoaffinity capture by magnetic beads or antibody functionalized columns/packing and immunoprecipitation. This technique is limited to EVs with known antigens (CD63, CD9, CD81, annexin, or EpCAM of the tetraspanin family). It allows separation of EVs bearing these antigens from contaminating proteins and other vesicles. However, the heterogeneity of EV produced by cells limits the efficacy of this approach [25 ]. Studies have revealed that there are no common proteins expressed in large quantities on the surface of EVs derived from different sources [26 ]. Even vectors derived from cancer cells may be devoid of cancer specific EPCAM antigens. Thus, immunocapture can minimize protein and other contamination, but is often too specific for an independent platform (antigenic platform). In addition, while magnetic beads allow for flow cytometric sorting and other analysis of EVs, the separation process requires more than one day for optimal recovery.

Size-based ultrafiltration is a commercial size-based separation technique and the applied exosome separation is size-exclusion chromatography (SEC), such as IzonqEV. In SEC, a porous stationary phase is used to sort macromolecules and particulate matter according to size. Components in the sample that have small hydrodynamic radii can pass through the pores, resulting in late elution. The hydrodynamic radius of the large component including the exosomes is excluded from the pores. Since SEC is typically performed using gravity flow, the vesicle structure and integrity remains largely intact, and the biological activity of the exosomes is preserved. Also, SEC has excellent reproducibility. However, the current manual method of separation with qEV is not scalable, which limits its scalability in high-throughput applications. Another popular size-based exosome separation technique is ultrafiltration. The basic principle of ultrafiltration is not different from conventional membrane filtration, where the separation of suspended particles or polymers depends mainly on their size or molecular weight. Ultrafiltration is faster than ultracentrifugation and requires no special equipment. However, the use of force leads to large vesicle deformation and rupture, which can bias the results of downstream analysis [27-29 ]. Moreover, contamination of blood proteins (mainly albumin) remains a problem for SEC, even with chromatographic separation.

Although ultracentrifugation and rapid precipitation are currently the established methods for the isolation of exosomes, their low yield recovery and long isolation time make them unsuitable for diagnostic and therapeutic applications. Exosomes isolated by using differential ultracentrifugation often contain proteins and lipoproteins. Due to the complexity of biological samples, contamination from other extracellular vesicles with similar physicochemical and biochemical properties is inevitable. For example, there is a significant overlap in physical properties such as density and solubility between exosomes and non-exosomes EV. In contrast, size is a robust physical property currently used to distinguish exosomes from other EVs [30 ]. For example, most proteins and lipoproteins range in size from 2 to 35nm, while exosomes are typically in the 50 to 200nm range. Furthermore, differences in EV size have been shown to affect their recognition and capture by target cells [31 ].

Size-based SEC is likely to allow higher purity and yield, but fails to concentrate and fractionate exosomes. Size-based ultrafiltration is faster than ultracentrifugation and does not require special equipment. It also allows for simultaneous separation, concentration and fractionation. However, vesicle deformation, cleavage and fusion reduce yield and may skew the results of downstream analysis. In addition, ultrafiltration can lead to clogging and vesicle trapping, resulting in reduced membrane life and inefficient separation.

Current commercial size-based exosome separation technologies are unable to achieve high yields and purity while maintaining the ability to concentrate and fractionate. Thus, a high yield of size-based fractionation of isolated exosomes is required.

Disclosure of Invention

One embodiment described herein is a system for isolating exosomes, the system comprising: a first chamber; a second chamber; a membrane positioned between the first and second chambers, and the membrane comprising a first membrane surface facing and at least partially defining the first chamber, a second membrane surface facing and at least partially defining the second chamber, and a plurality of asymmetrically-shaped nanopores extending between the first and second membrane surfaces, wherein each nanopore comprises a first nanopore opening at the first membrane surface having a first diameter, and a second nanopore opening at the second membrane surface having a second diameter greater than the first diameter; a sample comprising exosomes located within the first chamber; and means for inducing fluid flow through the membrane from the first chamber to the second chamber by pressure driven flow, electroosmotic flow, centrifugal force, or a combination thereof. In one aspect, the first chamber includes a wall opposite the first membrane, the wall including one or more baffling fluids (baffles).

Another embodiment described herein is a system for isolating exosomes, the system comprising: a first chamber; a second chamber; a membrane located between the first and second chambers, and the membrane comprising a first membrane surface facing and at least partially defining the first chamber, a second membrane surface facing and at least partially defining the second chamber, and a plurality of asymmetrically-shaped nanopores extending between the first and second membrane surfaces, wherein each nanopore comprises a first nanopore opening at the first membrane surface having a first diameter, and a second nanopore opening at the second membrane surface having a second diameter greater than the first diameter; wherein the first chamber comprises a wall opposite the first membrane, the wall comprising one or more baffling fluids; a sample comprising exosomes located within the first chamber; and means for inducing fluid flow through the membrane from the first chamber to the second chamber by pressure driven flow, electroosmotic flow, centrifugal force, or a combination thereof. In one aspect, the first film surface is coated with a magnetic alloy. In another aspect, the first diameter is between about 10nm and about 200 nm. In another aspect, the second diameter is less than about 2 μm. In another aspect, the membrane is formed from one or more materials including one or more of polyethylene terephthalate (PET), Polycarbonate (PC), polypropylene (PP), Polyimide (PI), or Polyethersulfone (PES). In another aspect, the system further includes a third chamber and a filter positioned between the third chamber and the first chamber, the filter including a first filter surface facing and at least partially defining the third chamber, a second filter surface facing and at least partially defining the first chamber, and a plurality of filter apertures extending between the first and second filter surfaces. In another aspect, each filter pore has a diameter of 200 nanometers to 5 micrometers. In another aspect, the filter is formed from one or more materials including polyethylene terephthalate (PET), Polycarbonate (PC), polypropylene (PP), Polyimide (PI), and Polyethersulfone (PES). In another aspect, the system further comprises a fourth chamber and a second membrane positioned between the fourth chamber and the second chamber, the second membrane comprising a first membrane surface facing and at least partially defining the second chamber and a second membrane surface facing and at least partially defining the fourth chamber; wherein the second film is a film as described herein; and wherein the first film surface is coated with a magnetic alloy. In another aspect, the means for inducing fluid flow produces a flow rate of between about 0.01 ml/hr and about 1000 ml/hr. In another aspect, the device that induces fluid flow generates a pressure less than about 1 atm. In another aspect, the means for inducing fluid flow comprises a syringe pump, an electroosmotic pump, a micropump, a centrifuge, or a combination thereof. In another aspect, the sample is applied to the membrane or the filter perpendicularly or tangentially. In another aspect, the magnetic alloy is nickel-iron, samarium-cobalt, aluminum-nickel-cobalt, or neodymium-iron-boron. In another aspect, the exosomes are bound to probes coupled to magnetic beads. In another aspect, the probe is an antibody.

Another embodiment described herein is a method for isolating exosomes, the method comprising: providing a system as described herein, and inducing a fluid flow through the membrane from the first chamber to the second chamber, whereupon the exosomes are isolated in the second chamber.

Another embodiment described herein is an exosome isolated using the method described herein.

Another embodiment described herein is a method for isolating exosomes, the method comprising: providing a system, the system comprising: a first chamber; a second chamber; a third chamber; a membrane positioned between the first and second chambers, and the membrane comprising a first membrane surface facing and at least partially defining the first chamber, a second membrane surface facing and at least partially defining the second chamber, and a plurality of asymmetrically-shaped nanopores extending between the first and second membrane surfaces, wherein each nanopore comprises a first nanopore opening at the first membrane surface having a first diameter, and a second nanopore opening at the second membrane surface having a second diameter greater than the first diameter; a filter positioned between the third chamber and the first chamber, the filter including a first filter surface facing and at least partially defining the third chamber, a second filter surface facing and at least partially defining the first chamber, and a plurality of filter pores extending between the first and second filter surfaces; and means for inducing fluid flow through the filter from the third chamber to the first chamber and through the membrane from the first chamber to the second chamber by pressure driven flow, electroosmotic flow, centrifugal force, or a combination thereof; introducing a sample comprising exosomes into the third chamber; inducing fluid flow through the filter and the membrane from the third chamber to the first chamber and from the first chamber to the second chamber whereupon the exosomes pass through the filter and are separated in the second chamber. In one aspect, the system further comprises a fourth chamber and a second membrane located between the fourth chamber and the second chamber, the second membrane comprising a first membrane surface facing and at least partially defining the second chamber and a second membrane surface facing and at least partially defining the fourth chamber; wherein the second film is a film as described herein; and wherein the first film surface is coated with a magnetic alloy. In another aspect, the first film surface is coated with a magnetic alloy. In another aspect, the exosome-containing sample comprises one or more of a cell culture supernatant, a sample obtained from an animal subject, or an apoplastic fluid from a plant. In another aspect, the sample obtained from the animal subject comprises one or more of blood, plasma, tears, serum, urine, sputum, pleural effusion, or ascites. In another aspect, the first diameter is between about 10nm and about 200 nm. In another aspect, the second diameter is less than about 2 μm. In another aspect, the membrane is formed from one or more materials including polyethylene terephthalate (PET), Polycarbonate (PC), polypropylene (PP), Polyimide (PI), or Polyethersulfone (PES). In another aspect, each filter pore has a diameter of 200 nanometers to 5 micrometers. In another aspect, the filter is formed from one or more materials including polyethylene terephthalate (PET), Polycarbonate (PC), polypropylene (PP), Polyimide (PI), or Polyethersulfone (PES). In another aspect, the first chamber includes a wall opposite the first membrane, the wall including one or more baffling bodies. In another aspect, the means for sample flow produces a flow rate of between about 0.01 ml/hr and about 1000 ml/hr. In another aspect, the device that induces fluid flow generates a pressure less than about 1 atm. In another aspect, the means for inducing fluid flow comprises an injection pump, an electroosmotic pump, a micropump, a centrifuge, or a combination thereof. In another aspect, the sample is applied to the filter perpendicularly or tangentially. In another aspect, the magnetic alloy is nickel-iron, samarium-cobalt, aluminum-nickel-cobalt, or neodymium-iron-boron. In another aspect, the exosomes are bound to probes coupled to magnetic beads. In another aspect, the probe is an antibody.

Another embodiment described herein is an exosome isolated using any of the methods described herein.

Drawings

FIG. 1 physical characteristics of different EV subtypes. Nanoparticles carrying biomarkers such as proteins and RNA: different nanoparticles are from different cells or from different parts of the same cell. Some are formed only during acute stress. Thus, by ranking them, one can gain insight into the source of their molecular load. They vary in size and specific density. Many nanoparticle separation techniques are based on these differences.

Fig. 2A shows a schematic diagram of an ANM manufacturing process.

Fig. 2B shows a Scanning Electron Microscope (SEM) image of the tip side before protocol optimization.

Fig. 2C shows an SEM image of the tip side after the protocol optimization.

Fig. 2D shows SEM images of the tip and base sides of 10nm symmetric nanopore membrane, 10nm ANM, and 20nm ANM.

Fig. 3 shows a tangential flow ANM filtration setup.

Fig. 4A and 4B are diagrams of a tangential flow ANM filtration prototype.

Figure 4C shows a schematic of the baffle design.

Fig. 4D is a 3D printed membrane holder with a folded fluid design.

Fig. 5A shows EV amounts and pressures for separations using different membranes with different degrees of pore asymmetry.

Figure 5B shows the pore size distribution before (left) and after separation using ANM (middle) and cylindrical nanopore membrane (right).

Fig. 6A is an estimate of the pressure exerted by an EV using a different separation method.

Fig. 6B and 6C are comparisons of EV amounts separated using different separation methods.

Figure 6D shows SEM images of isolated EVs and Western blot analysis of exosome marker CD 63.

Fig. 7A shows a schematic of exosome separation using a tangential flow ANM nanofiltration device.

Figure 7B shows the protein concentration in the through-flow as a function of the volume of wash buffer pumped through the device.

Fig. 7C shows the size distribution before and after separation.

FIG. 7D is a comparison of extraction yields between ANM and other commercial technologies (ExoQuick-TC, qEV) and UC. The use of tangential flow increases the isolation yield to 90%.

FIG. 8 shows size-based EV ranking using 200nm ANM (FIG. 8A) and 100nm ANM (FIG. 8B).

Fig. 9A is a workflow for immunocapture using Magnetic Nanopore Membranes (MNMs).

Fig. 9B is a diagram of MNM made by conventional rotary stirring electroplating (left) and custom stirring apparatus (right).

Figure 9C shows the device for running the nano-immunocapture experiments.

FIG. 9D shows an SEM image of a NiFe layer deposited with the original plating solution.

FIG. 9E shows an SEM image of a NiFe layer deposited with a sugarless finish plating solution.

Fig. 10A is an SEM image of Magnetic Nanobeads (MNBs) captured on the membrane surface.

Fig. 10B shows recovery of MNB under different conditions.

Fig. 11A shows the workflow of MNM capture after ANM isolation and direct MNM capture.

Fig. 11B shows the yields of MNM capture and direct MNM capture after ANM isolation.

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For example, any nomenclature used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein is those well known and commonly used in the art. In the event of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, but methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention.

As used herein, terms such as "comprising," including, "" having, "and the like, refer to" comprising. The present disclosure also contemplates other embodiments "comprising," "consisting of," and "consisting essentially of," whether or not explicitly stated.

As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well as the plural forms thereof, unless the context clearly indicates otherwise. Furthermore, unless otherwise specified, the reference to "one or more" is intended to mean "one or more".

The term "or" as used herein may be conjunctive or disjunctive.

The term "substantially" as used herein means to a substantial or significant extent, but not completely.

As used herein, the term "about" or "approximately" when applied to one or more values of interest refers to a value that is similar to the indicated reference value, or within the tolerance of the particular value, as determined by one of ordinary skill in the art, which will depend in part on the manner in which the value is measured or determined, e.g., the limitations of the measurement system. In one aspect, the term "about" refers to any value, including integer and fractional portions that vary by up to ± 10% of the value modified by the term "about". Alternatively, "about" can mean within 3 or more than 3 standard deviations, according to practice in the art. Alternatively, for example, with respect to a biological system or process, the term "about" can mean within an order of magnitude, in some embodiments within 5-fold of the value, and in some embodiments within 2-fold of the value.

All ranges disclosed herein are inclusive of the two endpoints being discrete values and all integers and fractions specified within the range. For example, a range of 0.1-2.0 includes 0.1, 0.2, 0.3, 0.4. If an endpoint is modified by the term "about," the stated range is extended by up to ± 10% variation or within 3 or more standard deviations of any value within the range, including the endpoint. The symbols "to" as used herein mean "about".

The term "exosome" refers to a cell-derived vesicle having a diameter of between about 20-250nm, for example between 40-210nm, for example, a diameter of about 50nm, 60nm, 70nm, 80nm, 90nm, 100mm, 110nm, 120nm, 130nm, 150nm, 160nm, 170nm, 180nm, 190nm or 200 nm. Exosomes may be isolated from any suitable biological sample of a mammal, including, but not limited to, whole blood, serum, plasma, urine, saliva, breast milk, cerebrospinal fluid, amniotic fluid, ascites, bone marrow, and cultured mammalian cells (e.g., immature dendritic cells (wild-type or immortalized), induced and non-induced pluripotent stem cells, fibroblasts, platelets, immune cells, reticulocytes, tumor cells, mesenchymal stem cells, satellite cells, hematopoietic stem cells, pancreatic stem cells, white and beige preadipocytes, and the like). As will be appreciated by those skilled in the art, the cultured cell sample is in a cell-appropriate medium (using exosome-free serum). Exosomes include specific surface markers not present in other vesicles, including surface markers such as tetraspanin proteins, e.g., CD9, CD37, CD44, CD53, CD63, CD81, CD82, and CD 151; targeting or adhesion markers, such as integrin, ICAM-1, EpCAM and CD 31; membrane fusion markers, such as annexin, TSG101, ALIX; and other exosome transmembrane proteins such as Rab5b, HLA-G, HSP70, LAMP2 (lysosomal associated membrane protein) and LIMP (lysosomal integral membrane protein). Exosomes may also be obtained from non-mammalian or cultured non-mammalian cells. Since the molecular mechanisms involved in exosome biogenesis are thought to be evolutionarily conserved, exosomes from non-mammalian sources include surface markers that are isoforms of mammalian surface markers, such as the isoforms of CD9 and CD63, which distinguish them from other cellular vesicles. The term "non-mammalian" is intended to include, for example, exosomes from microorganisms such as bacteria, flies, worms, plants, fruits/vegetables (e.g., corn, pomegranate) and yeast.

As used herein, a "sample" may refer to any sample in which the presence and/or level of a target is to be detected or determined, or any sample comprising exosomes or components thereof as described herein. The sample may comprise a liquid, solution, emulsion or suspension. The sample may comprise any plant fluid or tissue, such as an apoplastic fluid, any biological fluid or tissue, such as blood, whole blood, blood fractions such as plasma and serum, muscle, interstitial fluid, sweat, saliva, urine, tears, synovial fluid, bone marrow, cerebrospinal fluid, nasal secretions, sputum, amniotic fluid, bronchoalveolar lavage fluid, gastric lavage fluid, vomit, stool, lung tissue, peripheral blood mononuclear cells, total leukocytes, lymph node cells, spleen cells, tonsillar cells, cancer cells, tumor cells, bile, pleural effusion, ascites, digestive juices, skin, or combinations thereof. The sample may be used directly with a sample obtained from a subject or may be pre-treated, e.g., filtered, distilled, extracted, concentrated, centrifuged, inactivated interfering components, added with reagents, etc., to modify the characteristics of the sample in some manner as discussed herein or otherwise known in the art.

The term "subject" as used herein refers to an animal. Typically, the animal is a mammal. Subjects also refer to, for example, primates (e.g., humans, male or female; infants, adolescents or adults), pigs, cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice, fish, birds, and the like. In one embodiment, the subject is a human.

Described herein is a size-based Asymmetric Nanopore Membrane (ANM) filtration technique for efficient exosome separation, concentration and fractionation. The ANM technique utilizes an asymmetric etching technique for commercial ion-track membranes to produce tapered nanopores, which can range from 10nm to 200nm on the tip side and up to 2 microns on the base side. Track-etched membranes with asymmetrically shaped pores (as opposed to the more conventional cylindrical or irregularly shaped pores in ultrafiltration membranes) offer important advantages for exosome-separation applications. A key advantage of a symmetric pore shape compared to a similar cylindrical pore membrane is that the pressure/force applied to drive the sample through the filter membrane at the same flux is drastically reduced by 200-. This significant reduction in applied pressure prevents exosome deformation, lysis and fusion, thereby significantly increasing yield (up to 90%), while retaining the other advantages of size-based ultrafiltration. Furthermore, the chances of clogging and vesicle trapping are significantly reduced compared to similar cylindrical pore membranes due to the dramatic increase in transport rates through the membrane. This new pore geometry design allows for high yield and high throughput, and allows for a capture design. The trapping design allows for the concentration of exosomes in a specific size range and separation from larger and smaller fragments, molecules and EVs. The concentration factor can be as high as 100. Importantly, the capture design allows for washing of the captured exosomes with a rinse buffer to remove all contaminants, including abundant proteins. It also provides higher throughput, yield, sample purity and concentration factor, and more accurate size classification than existing products.

The ANM is high throughput because the tapered geometry reduces the flow shear rate. Lower shear rates also minimize nanoparticle loss due to fragmentation. The result is a high yield and high throughput platform that can separate exosomes (approximately 50 to 200nm in size) from proteins, RNPs, HDL and LDL. The tapered nanopore is fabricated by asymmetric wet etching of an ion track membrane without a dielectric coating. The technique has been validated with cell culture medium/supernatant and plasma samples. ANM showed much higher yields and throughput compared to precipitation techniques (exotick), ultracentrifugation, size exclusion (qEV) and column adsorption (miReasy). Throughput is particularly high, requiring about 1 hour for about 1mL of cell culture medium and about 300 μ L of plasma, compared to several days for other techniques. qEV have comparable fluxes, but it is not graded.

The isolated and purified exosomes may be cleaved by mechanical, thermal or chemical means to release their molecular biomarker cargo for quantification. Such quantification can be accomplished using a number of techniques, including ANM miRNA quantification techniques that do not suffer from PCR amplification bias. The AMN filtration technique allows complete EV and protein separation due to the presence of a 30nm asymmetric nanopore filter and the step of adding buffer to wash the exosomes trapped between the two membranes. Thus, high recovery efficiency can be achieved without sacrificing protein removal. Furthermore, the method does not require timing (timing), which introduces significant complexity in the separation process and reduces throughput. The ANM technique simultaneously separates and concentrates EV from any volume up to 5mL, up to 4mL, up to 3mL, up to 2mL, up to 1mL, up to 500. mu.L, or up to 300. mu.L. The concentration factor can be as high as 10 to 100. The nanopore technology of the present invention allows all exosomes of a size larger than the tip size of the pore to have the same separation efficiency, and therefore introduces less bias in the separation step. The AMN technique allows for precise control of pore size so that size-based fractionation can be performed in the 30-200nm range (by using different nanopore membrane modules with different pore sizes).

ANM consists of a membrane support and a commercial micropump or syringe pump. The pump may be installed in a dedicated instrument, or the consumer may use their own syringe pump in their laboratory. One embodiment includes the ANM and its holder, which may be discarded after each use. The ANM may be made from a polycarbonate track etch film that is initially irradiated to produce the desired ion tracks and then etched to develop the tracks into holes. The track irradiation step enables mass production. The etching process includes chemical etching and dry etching, which are also easy to scale up.

One embodiment described herein is a system for isolating exosomes, the system comprising: a first chamber; a second chamber; a membrane positioned between the first and second chambers and comprising a first membrane surface facing and at least partially defining the first chamber, a second membrane surface facing and at least partially defining the second chamber, and a plurality of asymmetrically-shaped nanopores extending between the first and second membrane surfaces, wherein each nanopore comprises a first nanopore opening having a first diameter at the first membrane surface and a second nanopore opening having a second diameter at the second membrane surface that is greater than the first diameter; a sample comprising exosomes located within the first chamber; and means for inducing fluid flow through the membrane from the first chamber to the second chamber by pressure driven flow, electroosmotic flow, centrifugal force, or a combination thereof. In one aspect, the first chamber includes a wall opposite the first membrane, the wall including one or more baffling fluids. In some embodiments, there may be at least 1, at least 2, at least 3, at least 4, at least 5 baffling fluids. In other embodiments, there may be up to 1000, up to 900, up to 800, up to 700, up to 600, up to 500, up to 400, up to 300, up to 200, up to 100, up to 50, or up to 25 folds. The baffling fluid may be made of fiberglass, plastic, composite materials, or other materials. In some embodiments, the fluid may be made of Polycarbonate (PC), Polystyrene (PS), polyethylene terephthalate (PET), polyvinyl chloride (PVC), SU-8 photoresist and Polyimide (PI), Polydimethylsiloxane (PDMS), silicon, or glass. In a particular embodiment, the baffling fluid may be made of Polymethylmethacrylate (PMMA). The baffle may be shaped as a cube, triangular prism, rectangle, cone, or curved, saw tooth, corrugated, or L-shaped slat, have a combination of these shapes, or be otherwise configured. The baffle geometry may be triangular, wedge, crescent, etc. They may exhibit a regular or staggered pattern, including a herringbone pattern. In a particular embodiment, the baffle may be a cube or triangular prism. The height of the baffling fluid can be from about 15 μm to about 3mm, about 20 μm to about 2mm, about 25 μm to about 2mm, about 30 μm to about 2mm, about 35 μm to about 1mm, about 40 μm to about 1mm, or about 45 μm to about 1 mm. The baffling fluid may be spaced apart by about 25 μm to about 7mm, about 50 μm to about 6mm, about 100 μm to about 5mm, about 100 μm to about 4mm, about 100 μm to about 3mm, about 100 μm to about 2mm, about 100 μm to about 1mm, about 125 μm to about 5mm, or about 150 μm to about 5 mm. The size, number and spacing of the baffling fluid can be varied and selected to provide the desired sample flow dispersion, course and rate for a particular application or particle to be separated. In some embodiments, each or a particular baffle has a gap formed all the way around them at the top and/or bottom, at one or both sides. In addition, the baffling fluid may be arranged in an array having a regular pattern or an irregular arrangement. And some of the baffling fluid may be larger than other baffling fluids. Previously, ultrafiltration baffles were placed directly on the membrane to create a vortex that disrupts the filter cake. However, the vortex flow also reduces the filtration rate. The present disclosure places baffles on the channel surface opposite the membrane without creating turbulence. The alignment and spacing of the baffling fluid depends on various factors such as the size range of the nanoparticles, diffusivity in a particular medium, membrane thickness, etc., and can be dictated by the diffusion timescale of the polarizing layer, normal and tangential flow rates, and the length of the entrance segment of the fluid flow. The baffles create an upward lifting force to break up the filter cake before it has sufficiently accumulated.

Another embodiment described herein is a system for isolating exosomes, the system comprising: a first chamber; a second chamber; a membrane positioned between the first and second chambers, and the membrane comprising a first membrane surface facing and at least partially defining the first chamber, a second membrane surface facing and at least partially defining the second chamber, and a plurality of asymmetrically-shaped nanopores extending between the first and second membrane surfaces, wherein each nanopore comprises a first nanopore opening at the first membrane surface having a first diameter, and a second nanopore opening at the second membrane surface having a second diameter greater than the first diameter; wherein the first chamber comprises a wall opposite the first membrane, the wall comprising one or more baffling fluids; is located in the first chamberA sample comprising exosomes of (a); and means for inducing fluid flow through the membrane from the first chamber to the second chamber by pressure driven flow, electroosmotic flow, centrifugal force, or a combination thereof. In one aspect, the first film surface is coated with a magnetic alloy. In another aspect, the system may further comprise a fourth chamber and a second membrane positioned between the fourth chamber and the second chamber, the second membrane comprising a first membrane surface facing and at least partially defining the second chamber and a second membrane surface facing and at least partially defining the fourth chamber. The second film may be a film as described herein (e.g., ANM), and the first film surface of the film may be coated with a magnetic alloy. In another aspect, the magnetic alloy is nickel-iron, samarium-cobalt, aluminum-nickel-cobalt, nickel-iron-chromium, iron-chromium-cobalt, or neodymium-iron-boron. In another aspect, the exosomes are bound to probes coupled to magnetic beads. The magnetic beads can be any of a wide variety of shapes, such as spherical, substantially spherical, egg-shaped, disk-shaped, cubic, and other three-dimensional shapes. The magnetic beads can be made using a wide variety of materials including, for example, resins and polymers. The magnetic beads may be of any suitable size, including, for example, microbeads, microparticles, nanobeads, and nanoparticles. The magnetic beads may comprise magnetically responsive material which may constitute substantially all of the beads or only one component of the beads. The remainder of the bead may include a polymeric material, a coating, and a moiety that allows attachment of assay reagents, and the like. Examples of suitable magnetic beads include flow cytometry microbeads, polystyrene microparticles and nanoparticles, functionalized polystyrene microparticles and nanoparticles, coated polystyrene microparticles and nanoparticles, silica microbeads, fluorescent microspheres and nanospheres, functionalized fluorescent microspheres and nanospheres, coated fluorescent microspheres and nanospheres, color-dyed microparticles and nanoparticles, magnetic microparticles and nanoparticles, superparamagnetic microparticles and nanoparticles (e.g.,

particles, available from Dynal Bead Based Separations (Invitrogen Group), Carlsbad, Calif) Fluorescent microparticles and nanoparticles, coated magnetic microparticles and nanoparticles, ferromagnetic microparticles and nanoparticles, coated ferromagnetic microparticles and nanoparticles, and other magnetic beads known in the art. In another aspect, the probe is an antibody. The antibody may bind to a surface marker on the exosome. In particular, the antibodies can bind CD9, CD31, CD37, CD44, CD53, CD63, CD81, CD82 and CD151, integrins, ICAM-1, EpCAM, annexin, TSG101, ALIX, Rab5b, HLA-G, HSP70, LAMP2, LIMP, other known exosome surface markers, or a combination thereof. In another aspect, the first diameter may be between about 5nm to about 300nm, about 5nm to about 200nm, about 10nm to about 300nm, about 10nm to about 200nm, about 10nm to about 150nm, about 10nm to about 100nm, about 10nm to about 50nm, about 20nm to about 300nm, about 20nm to about 200nm, about 20nm to about 100nm, or about 50nm to about 200 nm. In one particular aspect, the first diameter may be between about 10nm to about 200 nm. The second diameter may be less than about 5 μm, less than about 4 μm, less than about 3 μm, less than about 2 μm, less than about 1 μm, or less than about 0.5 μm. In one particular aspect, the second diameter can be less than about 2 μm. The nanopores may be arranged in an array having a regular pattern or an irregular arrangement. And some of the baffling fluid may be larger than other baffling fluids. In another aspect, the membrane is formed from one or more materials including one or more of polyethylene terephthalate (PET), Polycarbonate (PC), polypropylene (PP), Polyimide (PI), or Polyethersulfone (PES). In another aspect, the system further includes a third chamber and a filter positioned between the third chamber and the first chamber, the filter including a first filter surface facing and at least partially defining the third chamber, a second filter surface facing and at least partially defining the first chamber, and a plurality of filter apertures extending between the first and second filter surfaces. In another aspect, the diameter of each filter pore can be 150 nanometers to 6 microns, 150 nanometers to 5 microns, 200 nanometers to 4 microns, 200 nanometers to 3 microns, 200 nanometers to 2 microns, 200 nanometers to 1 micron, 300 nanometers to 5 microns, 400 nanometers to 5 microns, 500 nanometers to 1 micron, in another aspect, the diameter of each filter pore can be 150 nanometers to 6 microns, 150 nanometers to 5 microns, 200 nanometers to 1 micron, 300 nanometers to 5 microns, 400 nanometers to 5 microns, 500 nanometers to 5 micronsNano to 5 microns, 600 nano to 5 microns, 700 nano to 5 microns, 800 nano to 5 microns, 900 nano to 5 microns, or 1000 nano to 5 microns. In another aspect, the filter is formed from one or more materials including polyethylene terephthalate (PET), Polycarbonate (PC), polypropylene (PP), Polyimide (PI), and Polyethersulfone (PES). In another aspect, the means for inducing fluid flow produces a flow rate of between about 0.01 ml/hr and about 1000 ml/hr, about 0.01 ml/hr and about 900 ml/hr, about 0.01 ml/hr and about 800 ml/hr, about 0.01 ml/hr and about 700 ml/hr, about 0.01 ml/hr and about 600 ml/hr, about 0.01 ml/hr and about 500 ml/hr, about 0.01 ml/hr and about 400 ml/hr, about 0.01 ml/hr and about 300 ml/hr, about 0.01 ml/hr and about 200 ml/hr, about 0.01 ml/hr and about 100 ml/hr, about 0.05 ml/hr and about 1000 ml/hr, about 0.1 ml/hr and about 1000 ml/hr, from about 0.2 ml/hr to about 1000 ml/hr, from about 0.3 ml/hr to about 1000 ml/hr, from about 0.4 ml/hr to about 1000 ml/hr, or from about 0.5 ml/hr to about 1000 ml/hr. In another aspect, the device that induces fluid flow generates a pressure less than about 0.3atm, less than about 0.4atm, less than about 0.5atm, less than about 1atm, less than about 1.1atm, less than about 1.2atm, less than about 1.3atm, less than about 1.4atm, less than about 1.5 atm. In particular, the device inducing fluid flow generates a pressure less than about 1 atm. In another aspect, the means for inducing fluid flow comprises a syringe pump, an electroosmotic pump, a micropump, a centrifuge, or a combination thereof. In another aspect, the sample is applied perpendicularly or tangentially to the membrane or the filter.

Another embodiment described herein is a method for isolating exosomes, the method comprising: providing a system as described herein, and inducing a fluid flow from the first chamber to the second chamber through the membrane prior to separating the exosomes in the second chamber.

Another embodiment described herein is an exosome isolated using the method described herein.

Another embodiment described herein is a method for isolating exosomes, the method comprising: providing a system, the system comprising: a first chamber; a second chamber; a third chamber; a membrane located between the first and second chambers, and the membrane comprising a first membrane surface facing and at least partially defining the first chamber, a second membrane surface facing and at least partially defining the second chamber, and a plurality of asymmetrically-shaped nanopores extending between the first and second membrane surfaces, wherein each nanopore comprises a first nanopore opening at the first membrane surface having a first diameter, and a second nanopore opening at the second membrane surface having a second diameter greater than the first diameter; a filter positioned between the third chamber and the first chamber, the filter including a first filter surface facing and at least partially defining the third chamber, a second filter surface facing and at least partially defining the first chamber, and a plurality of filter pores extending between the first and second filter surfaces; and means for inducing fluid flow through the filter from the third chamber to the first chamber and through the membrane from the first chamber to the second chamber by pressure driven flow, electroosmotic flow, centrifugal force, or a combination thereof; introducing a sample comprising exosomes into the third chamber; inducing fluid flow through the filter and the membrane from the third chamber to the first chamber and from the first chamber to the second chamber whereupon the exosomes pass through the filter and are separated in the second chamber. In one aspect, the exosome-containing sample comprises one or more of a cell culture supernatant, a sample obtained from an animal subject, or an apoplastic fluid from a plant. In another aspect, the sample obtained from the animal subject comprises one or more of blood, plasma, tears, serum, urine, sputum, pleural effusion, or ascites. In another aspect, the first diameter is between about 10nm and about 200 nm. In another aspect, the second diameter is less than about 2 μm. In another aspect, the membrane is formed from one or more materials including polyethylene terephthalate (PET), Polycarbonate (PC), polypropylene (PP), Polyimide (PI), or Polyethersulfone (PES). In another aspect, each filter pore has a diameter of 200 nanometers to 5 micrometers. In another aspect, the filter is formed from one or more materials including polyethylene terephthalate (PET), Polycarbonate (PC), polypropylene (PP), Polyimide (PI), or Polyethersulfone (PES). In another aspect, the first chamber includes a wall opposite the first membrane, the wall including one or more baffling fluids. In another aspect, the means for sample flow produces a flow rate of between about 0.01 ml/hr and about 1000 ml/hr. In another aspect, the device that induces fluid flow generates a pressure less than about 1 atm. In another aspect, the means for inducing fluid flow comprises a syringe pump, an electroosmotic pump, a micropump, a centrifuge, or a combination thereof. In another aspect, the sample is applied to the filter perpendicularly or tangentially.

Another embodiment described herein is an exosome isolated using any of the methods described herein.

It will be apparent to one of ordinary skill in the relevant art that appropriate modifications and adaptations to the compositions, formulations, methods, processes and applications described herein may be made without departing from the scope of any embodiment or aspect thereof. The compositions and methods provided are exemplary and are not intended to limit the scope of any given embodiment. The various embodiments, aspects and options disclosed herein may all be combined in any variation or new edition. The scope of the compositions, formulations, methods and processes described herein includes all actual or potential combinations of the embodiments, aspects, options, examples and preferences described herein. The compositions, formulations, or methods described herein may omit any component or step, substitute for any component or step disclosed herein, or include any component or step disclosed elsewhere herein. The ratio of the mass of any component of any composition or formulation disclosed herein to the mass of any other component in the formulation, or to the total mass of other components in the formulation, is hereby disclosed as if they were expressly disclosed. To the extent that the meaning of any term in any patent or publication incorporated by reference conflicts with the meaning of the term used in the present disclosure, the meaning of the term or phrase in the present disclosure shall govern. Furthermore, the specification discloses and describes only exemplary embodiments. All patents and publications cited herein are incorporated herein by reference for their specific teachings.

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Examples

Example 1

General procedure

Asymmetric Nanopore Membrane (ANM)

Track-etched films are prepared by a track-etching technique based on the irradiation of a material with fast heavy ions followed by chemical etching. The aperture size can be controlled by the etching time and the number of ions per unit area determines the number of damaged tracks and thus the number of apertures. Polycarbonate membranes of this type have cylindrical pores with diameters ranging from as small as 10nm to as large as 20 μm and a pore density as high as 5X 10 ⁸ cm ^-2 Commercially available. The study used 30nm PC membranes with a thickness of 6 μm from Sigma (Whatman Nuclepore track etched membranes). The received film had a cylindrical pore shape and a pore density of 5X 10 ⁸ cm ^-2 . The pore size and density of the received membrane was confirmed by SEM (fig. 2C). Asymmetric nanopores are formed by simple O ₂ A plasma etch process is generated on one side of the received track film (fig. 2A). A cylindrical pore membrane 25mm in diameter was placed on a silicon wafer (500 μm thick). One surface of these films appeared glossy while the opposite surface appeared rough. The film was placed on the silicon wafer with the rough surface facing up. A 2.5cm x 2.5cm PMMA plate with a hole of 21mm diameter cut into it was placed on top of the film and the PMMA plate was attached to the silicon wafer using Kapton tape. The hole limits the exposure of the membrane to O ₂ A region of plasma. O-etching Using a commercial reactive ion etching System (Oxford plasma Pro System, type RIE 100) ₂ And (4) plasma etching. The etching conditions were as follows: o is ₂ Gas pressure 200Pa, gas flow rate 30 standard cm ³ min ^-1 And the power is 100W. As shown in fig. 2A and 2C, the plasma etching enlarges the hole diameter at the upper surface, but the hole diameter at the lower surface remains unchanged. In addition, plasma etching also reduces the thickness of the film.

Preparation of biological fluid samples

Whole blood samples were obtained from healthy patients and mice during fasting. For plasma samples, 2mL of whole blood was collected in Vacutainer tubes containing EDTA as an anticoagulant and centrifuged at 1900 × g (3000rpm) and 4 ℃ for 10 minutes to separate the plasma fraction. Fresh plasma samples (50-300 μ L) were immediately used for exosome-isolation experiments. Individual tissue cultures of different cell lines (LOX melanoma cells, PC3 mouse prostate cancer cells, MCF-7 breast cancer cells, OVCAR5 ovarian cancer cells) were grown to 90% confluence at 37 ℃, and cell culture supernatants were harvested and centrifuged at 2000 × g for 20 min.

Exosome isolation

Exosome separation was performed by direct-current nanofiltration using the prepared asymmetric nanopore membrane. The film was sealed in a homemade plastic film holder. The plastic housing is secured with metal screws and nuts and a plastic annular gasket provides a leak-free seal. The separation involves a size-based separation step and a washing step. The cell culture supernatant and diluted plasma sample (dilution factor: 40) were pre-filtered with 0.22 μm PES syringe filter and introduced continuously into the asymmetric nanopore membrane filtration device through a 5mL syringe using a syringe pump at a constant flow rate (5mL/h), followed by a 5mL 1 × PBS wash step. Concentrated exosomes were recovered from the fluid compartment (capacity-300 μ Ι _) next to the asymmetric nanopore membrane, and isolated EVs were then used for downstream physical characterization. When processing large volume and heterogeneous samples, exosome separations were also performed in tangential flow nanofiltration mode. Cake formation and high build-up pressure lead to exosome lysis and coalescence, especially when highly heterogeneous samples are filtered in large quantities. In the tangential flow nanofiltration assay, the feed stream passes parallel to the asymmetric nanopore membrane face, with a portion passing through the membrane (permeate) and the remainder (retentate) being recycled. The tangential flow ANM filtration platform is shown in fig. 3. The cell culture supernatant and diluted plasma sample (dilution factor: 40) were pre-filtered with a 0.22 μm PES syringe filter and introduced continuously by a syringe pump at a flow rate (10mL/h) while a peristaltic pump recirculates the retentate stream at a flow rate (40mL/h) to prevent formation of a restrictive layer, followed by a washing step up to 30mL of 1 XPBS. ANM flow chips were made by 3D printing chips with channel dimensions of 65(L) x 20(W) x 1(H) mm. A baffled tangential flow design is also introduced to better inhibit fouling and cake formation. These baffles are made on the top wall of the flow channel so that they are part of a flow chamber made of Polymethylmethacrylate (PMMA), as shown in fig. 4D. The shape of the fluid may resemble a cube or a triangular prism. The baffling fluid may range in height from about 25 μm to about 2mm and be spaced apart from about 100 μm to about 5 mm. The baffle design allows for different shear rates and polarizing layer lengths of the filter cake at the baffles and at the spacing between the baffles, as shown in fig. 4C. The difference in characteristic polarization length and shear rate of the filter cake causes it to break at the point of change. The two-dimensional baffling fluid may also induce turbulence in the system, thereby disrupting the filter cake. The inspiration for baffle design comes from the special filtering structure in filter-feeding (e.g., suspended matter feeding) fish that can significantly enhance the removal of restrictive clogging layers by inducing local vortices, as shown in fig. 3. The concentrated exosomes were recovered from the flow chip (capacity-2 mL) next to the asymmetric nanopore membrane.

MNM production

Briefly, 80nm Au was deposited onto one side of a 450nm track etched polycarbonate film (Whatman) using a thermal evaporator (Oerlikon Leybold 8-pocket e-beam) to provide a working electrode in a subsequent electrodeposition process. Then adding 200nm Ni ₈₀ Fe ₂₀ A thin film was electrodeposited on top of the Au thin film. A Ni electrode was used for the electrodeposition solution. Ni ₈₀ Fe ₂₀ The electrodeposition solution is prepared from 289g/L NiSO ₄ ·6H ₂ O、64g/L FeSO ₄ ·7H ₂ O、40g/L H ₃ BO ₃ 8.9 g/L5-sulfosalicylic acid dihydrate and 3 g/L1, 3, (6,7) -naphthalene trisulfonic acid trisodium salt hydrate. During electrodeposition, a deposition current<2.5mA/cm ² . The resulting MNM had an asymmetric geometry with a base diameter of about 450nm and a tip diameter of about 250 nm.

Isolation of exosomes using MNM

As detailed herein, exosomes were first isolated from mouse plasma using ANM based on their size. Immunoselection of exosomes was performed by performing positive selection using magnetic nanobeads (Miltenyi Biotec Inc.) recognizing the tetraspanin proteins CD9, CD63 or CD 81. These antibody-bearing magnetic nanobeads (20-30nm) were added to the sample (isolated exosomes) and incubated for 30 min at room temperature with shaking. The sample was then added to the reservoir of the MNM holder and the exosome sample was pumped at a flow rate of 1mL/h by applying pressure through a programmable syringe pump. The MNM holder was made by a computer controlled milling machine (Roland, monoFab SRM-20). Two annular neodymium magnets are placed on the top and bottom sides of the MNM holder, respectively, to provide a magnetic field to magnetize the MNM. When a sample solution is pumped through the chip, the magnetic nanoparticle-labeled exosomes are captured at the edges of the MNM's pores.

Characterization of isolated exosomes

The isolated exosome samples collected from the chip were then diluted-20-500 fold in 1 × PBS buffer. Nanoparticle tracking analysis was performed using Nanosight NS300(Nanosight, Wiltshire, UK). Five 60 seconds of video were recorded per sample, with the camera level and detection threshold set at 10.

Example 2

Preparation of ANM

The ion tracking membrane has uniform pore size and straight pores. For EV nanofiltration, they are better than commercial ultrafiltration membranes (e.g. PES membranes) with irregular pore geometry and non-specific binding. However, ion-track membranes have two major disadvantages: (1) fusion/clogging in nanopores and (2) filter cake formation when the membrane surface is not large enough for concentrated suspensions in plasma such as EV. The tapered pore geometry of the present disclosure prevents fusion and clogging of the nanopores. In addition, the tapered hole geometry allows for slight operating forces, low resistance to sample flow, and reduced fouling. It is based on ANM with uniform tip size and pore geometry produced by Reactive Ion Etching (RIE) of commercial ion track films, as shown in fig. 2A-2C. After calibrating the etch rate and optimizing all parameters (e.g., residual stress of the unetched nanopore membrane, resistance of the substrate silicon wafer, and spacing between the unetched membrane and the substrate), this novel etch scheme resulted in a success rate of greater than 90% for obtaining a uniform ANM with reproducible filtering performance. To rank EVs based on size, different ANMs with different pore cut-off sizes were developed. The new fabrication scheme enables fabrication of ANMs with tip pore sizes in the range of about 10nm to about 200 nm. FIG. 2C shows representative Scanning Electron Microscopy (SEM) images of 10nm and 20nm ANM. Precise pore cut-off size control of ANM (down to 10nm) can enable direct fractionation of ultra-small extracellular RNA vectors such as High Density Lipoprotein (HDL) and exosomes.

Example 3

Design of tangential flow ANM nanofiltration platform

A tangential flow ANM nanofiltration design was implemented to overcome filter cake formation. Two designs were developed: (1) a "dead-end" filtration cartridge, in which the feed stream is applied perpendicular to the membrane face and 100% of the fluid is passed through the membrane, and (2) a tangential flow filtration chip, in which the feed stream is applied parallel to the membrane face and a portion of the feed stream is passed through the membrane (permeate) while the remainder (retentate) can be recycled or fed to the next chip. Recycle streams may also be used for the dead-end design. To minimize filter cake formation, the membrane area is increased by attaching different cassettes or chips. Baffles were added to the top substrate of the tangential flow filtration chips. The channel height within the 3-D printing tangential flow chip was optimized to 1mm to reduce dead volume and allow high throughput (fig. 3). Flow guiding columns are made at the inlet and outlet of the channel to distribute the flow evenly. A portable instrument for tangential flow ANM filter chips was developed as shown in fig. 4A. The three-stage design shown in fig. 4D achieves a significant flux increase-after 30 x dilution, the flux of the media sample is 30mL/h and the plasma sample is 1mL plasma/h.

Example 4

Validation of ANM techniques

To highlight the unique advantages of ANM filtration, a study was conducted to demonstrate how the conical pore asymmetry of the ANM significantly reduces pumping pressure and clogging. By stopping the etching process at different time intervals, the hole geometry is adjusted from a cylinder to a perfect cone (fig. 5A). When the cone is fully formed, the pumping pressure drops significantly. For straight pore membranes, pressures are up to 150kPa, whereas for tapered nanopore membranes the pressure is only 20kPa — for ANM, the pressure is reduced by a factor of 7.5. Meanwhile, Nanoparticle Tracking Analysis (NTA) showed that the number of separated EVs doubled when using cone geometry. Careful examination of the particle size distribution in the retentate of the straight pore membrane (fig. 5B) revealed significant EV loss and the appearance of larger particles formed by fusion (represented by new peaks beyond 200 nm). For ANM, there is no such size distribution distortion, and the power-law size distributions of EVs before and after filtering are similar. Therefore, ANM was able to achieve high yields of more than 70%, which was consistently observed in different cell lines as well as plasma samples. This indicates that the ANM filtering technique is robust and can be used for a variety of cell and sample types. Furthermore, these results indicate that fusion results in most of the nanocarrier losses, and that the conical pore geometry of ANM minimizes such losses. Since the conical geometry also reduces the pressure gradient, a correlation between pressure and loss is established.

The ANM filtration technique was compared to other commonly used EV separation techniques. Fig. 6A and 6B show an estimation of the pressure exerted on the EV using different separation strategies and a yield comparison of the separated EVs, respectively. The Ultracentrifugation (UC) method separates EV's with high g-forces (-100000 g), which can be converted to an equivalent inertial pressure of 120 atm. In contrast, the pressures required for a 5mL/h flow rate for the straight-hole membrane and ANM are much lower, being about 1.5atm and 0.2atm, respectively. As shown in fig. 6B, the yield of UC process was lowest, while the EV number isolated by ANM was highest. This demonstrates that there is a negative correlation between yield and pressure. These results were also confirmed by Western blot analysis of the expression level of the isolated exosome protein biomarker (CD63) (fig. 6D). The high yield of EV isolated by ANM was found to be independent of the sample. Both CCM of MCF-7 breast cancer cells and EV isolation of human plasma showed consistent results (FIG. 6C). EV losses may be related to pressure, as pore geometry can increase pressure in addition to fusion, high pressure itself can cause fusion and lysis.

Example 5

High yield exosome separation with tangential flow ANM nanofiltration device

The exosomes were isolated from the heterogeneous sample using a tangential flow ANM nanofiltration device and the yield and purity of the isolated exosomes were characterized. To isolate exosomes, samples (cell culture supernatant or diluted plasma) were passed sequentially through 200nm ANM filtration cassettes at a flow rate of 5mL/h to filter out large EVs, then through 50nm ANM filtration cassettes to isolate and concentrate exosomes (fig. 7A). Tangential flow of buffer solution was introduced to prevent cake formation on the surface of the ANM and plugging at the nanopore tip (fig. 7A). The AMN filtration technique allows for complete EV and protein separation due to the presence of a 50nm asymmetric nanopore filter and an additional buffer washing step of exosomes trapped between the two membranes. Figure 7B shows the exponential decay of protein concentration in the through-flow as a function of wash buffer pumped through the device. Analysis of the fractions by NTA showed successful separation of the particle mixture (before separation) into concentrated 30-220nm EV (after separation; FIG. 7C). The exosome yield using ANM was-10-20 times higher than size exclusion chromatography (qEV) and precipitation technique (exotick-TC), 10 times higher than cylindrical nanopore membranes (fig. 7E). A 30-200nm small EV subset enriched in CD63 was isolated (referred to herein as the small EV or sEV fraction). This fraction is free of large EV, LDL and exosome particles as well as protein markers of classical exosomes. However, ApoA1 was detected in this sample, indicating that HDL particles (slightly less than 30nm) were still captured by ANM along with sEV. Purity will be further improved by eliminating HDL in the isolated exosome population using a baffled tangential flow design as described above. The baffled fluid creates a vortex in the tangential flow to prevent the formation of a filter cake that causes HDL capture. Two-stage ANM separation can also be used. Furthermore, there was no sEV observable in the pass stream, confirming the efficiency of capture sEV using the AMN technique. The AMN isolated sEV fraction was characterized in various tumor cell lines and sera.

To rank EVs into large EVs and sEV, the sample was first passed through a 200nm ANM filter cartridge to separate the larger EVs, and then through a 30nm ANM filter cartridge to separate sEV. Unlike the previous sEV separation experiment, the EV in the retentate from the 200nm ANM filtration step was also collected and corresponded to the large EV fraction. NTA analysis of the separated fractions showed successful fractionation of heterogeneous EVs into large EVs and sEV after separation (fig. 8A). In the large EV fraction, 90% of sEV has been removed. Purity will be further improved by optimizing the tangential flow rate and wash buffer to allow for better size separation. The use of 100nm ANM instead of 200nm ANM also allowed separation of EVs greater than 150nm (FIG. 8B).

Example 6

Development of an ANM-based immunocapture device

Immunocapture uses antibodies against different surface proteins. Traditional immunocapture methods use micron-sized magnetic beads and bulk magnets to pellet the beads. The low diffusivity of these large beads results in a long incubation time that may require more than 24 hours. Magnetic nanoscale-sized beads, on the other hand, can be captured in less than 1 hour. However, they are difficult to capture due to their paramagnetism. To solve this problem, a scheme of coating ANM with a nickel-iron alloy layer was developed to manufacture a Magnetic Nanopore Membrane (MNM) for rapid immunocapture of specific carriers, as shown in fig. 9A to 9E. The fabricated membranes are stable and can be easily integrated into microfluidic platforms for rapid separation of EV, LLP and RNP.

Recovery of Magnetic Nanobeads (MNBs) by MNM was tested with 30nm MNBs. Fig. 10A shows an SEM image of MNB captured near the pore entrance. Due to the high magnetic field gradient at the corners, a large fraction of particles are trapped at the edges of the hole. The NTA results show that the recovery of membranes with a pore size of 250nm is more than 95% at a flux of 1mL/h (FIG. 10B). The yield decreases with increasing pore size and flow. When macropores and high flow rates are used simultaneously, less than 20% of the nanobeads are collected with the membrane. The need for small pores in the MNM means that larger nanocarriers need to be removed before capture of the MNB, otherwise they can clog the MNM. Thus, this study highlights the benefits of a multi-stage design, where the upstream ANM module is integrated with the downstream MNB and MNM immunocapture modules.

Example 7

EV fractionation of magnetic Nanobeads/nanopore membranes (MNB/MNM) from plasma

The yield of isolation of EV subgroups with MNM was tested with healthy human plasma. Direct immunocapture of unpurified plasma samples and immunocapture of EV isolated and purified by ANM were studied (fig. 11A-11B). Briefly, 20 μ L of nanobeads with a set of exosome tetraspanin antibodies (CD9, CD63, CD81) were mixed with 1mL samples of the two. After incubation for 30 minutes at room temperature, the mixture was passed through MNM for 1 hour. NTA results showed that greater than 70% of the total EV was captured with the prepurified sample (fig. 11B). However, direct MNM immunocapture only achieved 20% recovery. This may be due to interference of other proteins in the original sample with the immunocapture. Currently, the entire immunocapture process takes 90 minutes. To further shorten the fractionation process, a new microfluidic device was developed that sequentially integrates both ANM and MNM in a continuous flow design. The raw sample is first filtered by the upstream ANM module and mixed with the MNB stream. The mixing channel used for stirring will serve to further accelerate the incubation process. The fully incubated mixture was passed through a downstream capture MNM module. This immunocapture technology was also developed for LLP and RNP, so the module could be a downstream stage.

Claims (39)

1. A system for isolating exosomes, the system comprising:

a first chamber;

a second chamber;

a membrane positioned between the first chamber and the second chamber, the membrane comprising a first membrane surface facing and at least partially defining the first chamber, a second membrane surface facing and at least partially defining the second chamber, and a plurality of asymmetrically-shaped nanopores extending between the first membrane surface and the second membrane surface, wherein each nanopore comprises a first nanopore opening at the first membrane surface having a first diameter, and a second nanopore opening at the second membrane surface having a second diameter greater than the first diameter;

a sample comprising exosomes located within the first chamber; and

means for inducing fluid flow through the membrane from the first chamber to the second chamber by pressure driven flow, electroosmotic flow, centrifugal force, or a combination thereof.

2. The system of claim 1, wherein the first chamber comprises a wall opposite the first membrane, the wall comprising one or more baffling fluids.

3. A system for isolating exosomes, the system comprising:

a first chamber;

a second chamber;

a membrane positioned between the first chamber and the second chamber, the membrane comprising a first membrane surface facing and at least partially defining the first chamber, a second membrane surface facing and at least partially defining the second chamber, and a plurality of asymmetrically-shaped nanopores extending between the first membrane surface and the second membrane surface, wherein each nanopore comprises a first nanopore opening at the first membrane surface having a first diameter, and a second nanopore opening at the second membrane surface having a second diameter greater than the first diameter;

wherein the first chamber comprises a wall opposite the first membrane, the wall comprising one or more baffling fluids;

a sample comprising exosomes located within the first chamber; and

means for inducing fluid flow through the membrane from the first chamber to the second chamber by pressure driven flow, electroosmotic flow, centrifugal force, or a combination thereof.

4. The system of any of claims 1-3, wherein the first film surface is coated with a magnetic alloy.

5. The system of any one of claims 1-3, wherein the first diameter is between about 10nm to about 200 nm.

6. The system of any one of claims 1-5, wherein the second diameter is less than about 2 μm.

7. The system of any one of claims 1-6, wherein the membrane is formed from one or more materials comprising one or more of polyethylene terephthalate (PET), Polycarbonate (PC), polypropylene (PP), Polyimide (PI), or Polyethersulfone (PES).

8. The system of any of claims 1-7, further comprising a third chamber and a filter positioned between the third chamber and the first chamber, the filter comprising a first filter surface facing and at least partially defining the third chamber, a second filter surface facing and at least partially defining the first chamber, and a plurality of filter pores extending between the first filter surface and the second filter surface.

9. The system of claim 8, wherein each filter pore is 200 nanometers to 5 microns in diameter.

10. The system of claim 8 or claim 9, wherein the filter is formed from one or more materials including polyethylene terephthalate (PET), Polycarbonate (PC), polypropylene (PP), Polyimide (PI), and Polyethersulfone (PES).

11. The system of any of claims 1-3 or 5-10, further comprising a fourth chamber and a second membrane positioned between the fourth chamber and the second chamber, the second membrane comprising a first membrane surface facing and at least partially defining the second chamber and a second membrane surface facing and at least partially defining the fourth chamber;

wherein the second film is the film of claim 21; and is provided with

Wherein the first film surface is coated with a magnetic alloy.

12. The system of any one of claims 1-11, wherein the means for inducing fluid flow produces a flow rate of between about 0.01 ml/hr and about 1000 ml/hr.

13. The system of any one of claims 1-12, wherein the means for inducing fluid flow generates a pressure of less than about 1 atm.

14. The system of any one of claims 1-13, wherein the means for inducing fluid flow comprises an injection pump, an electroosmotic pump, a micropump, a centrifuge, or a combination thereof.

15. The system of any one of claims 1-14, wherein the sample is applied to the membrane or the filter perpendicularly or tangentially.

16. The system of any one of claims 4-15, wherein the magnetic alloy is nickel-iron, samarium-cobalt, aluminum-nickel-cobalt, nickel-iron-chromium, iron-chromium-cobalt, or neodymium-iron-boron.

17. The system of any one of claims 4-16, wherein the exosomes are bound to probes coupled to magnetic beads.

18. The system of claim 17, wherein the probe is an antibody.

19.A method for isolating exosomes, the method comprising:

providing a system according to any of claims 1-18, and

inducing fluid flow through the membrane from the first chamber to the second chamber, thereby separating the exosomes in the second chamber.

20. An exosome isolated using the method of claim 19.

21. A method for isolating exosomes, the method comprising:

providing a system, the system comprising:

a first chamber;

a second chamber;

a third chamber;

a membrane positioned between the first chamber and the second chamber, the membrane comprising a first membrane surface facing and at least partially defining the first chamber, a second membrane surface facing and at least partially defining the second chamber, and a plurality of asymmetrically-shaped nanopores extending between the first membrane surface and the second membrane surface, wherein each nanopore comprises a first nanopore opening at the first membrane surface having a first diameter, and a second nanopore opening at the second membrane surface having a second diameter greater than the first diameter;

a filter positioned between the third chamber and the first chamber, the filter including a first filter surface facing and at least partially defining the third chamber, a second filter surface facing and at least partially defining the first chamber, and a plurality of filter pores extending between the first filter surface and the second filter surface; and

means for inducing fluid flow through the filter from the third chamber to the first chamber and through the membrane from the first chamber to the second chamber by pressure driven flow, electroosmotic flow, centrifugal force, or a combination thereof;

introducing a sample comprising exosomes into the third chamber;

inducing fluid flow through the filter and the membrane from the third chamber to the first chamber and from the first chamber to the second chamber whereupon the exosomes pass through the filter and are separated in the second chamber.

22. The system of claim 21, further comprising a fourth chamber and a second membrane positioned between the fourth chamber and the second chamber, the second membrane comprising a first membrane surface facing and at least partially defining the second chamber and a second membrane surface facing and at least partially defining the fourth chamber;

wherein the second film is the film of claim 21; and is

Wherein the first film surface is coated with a magnetic alloy.

23. The system of claim 21, wherein the first film surface is coated with a magnetic alloy.

24. The method of any one of claims 21-23, wherein the exosome-containing sample comprises one or more of a cell culture supernatant, a sample obtained from an animal subject, or an apoplastic fluid from a plant.

25. The method of any one of claims 21-24, wherein the sample obtained from the animal subject comprises one or more of blood, plasma, tears, serum, urine, sputum, pleural effusion, or ascites.

26. The method of any one of claims 21-25, wherein the first diameter is between about 10nm to about 200 nm.

27. The method of any one of claims 21-26, wherein the second diameter is less than about 2 μ ι η.

28. The method of any one of claims 21-27, wherein the membrane is formed from one or more materials comprising polyethylene terephthalate (PET), Polycarbonate (PC), polypropylene (PP), Polyimide (PI), or Polyethersulfone (PES).

29. The method of any one of claims 21-28, wherein each filter pore is 200 nanometers to 5 microns in diameter.

30. The method of any one of claims 21-29, wherein the filter is formed from one or more materials comprising polyethylene terephthalate (PET), Polycarbonate (PC), polypropylene (PP), Polyimide (PI), or Polyethersulfone (PES).

31. The method of any one of claims 21-30, wherein the first chamber comprises a wall opposite the first membrane, the wall comprising one or more baffling fluids.

32. The method of any one of claims 21-31, wherein the means for flowing the sample produces a flow rate of between about 0.01 ml/hr to about 1000 ml/hr.

33. The method of any one of claims 21-32, wherein the device that induces fluid flow generates a pressure less than about 1 atm.

34. The method of any one of claims 21-33, wherein the means for inducing fluid flow comprises an injection pump, an electroosmotic pump, a micropump, a centrifuge, or a combination thereof.

35. The method of any one of claims 21-34, wherein the sample is applied to the filter perpendicularly or tangentially.

36. The system of any one of claims 22-35, wherein the magnetic alloy is nickel-iron, samarium-cobalt, aluminum-nickel-cobalt, nickel-iron-chromium, iron-chromium-cobalt, or neodymium-iron-boron.

37. The system of any one of claims 22-36, wherein the exosomes are bound to probes coupled to magnetic beads.

38. The system of claim 37, wherein the probe is an antibody.

39. An exosome isolated using the method according to any one of claims 21-38.

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